Vitamin D is a well known vitamin which has many useful functions in mammals. It is activated by 25-hydroxylation in the liver and subsequently by 1-hydroxylation in the kidney. This stimulates intestinal calcium transport, the mobilization of calcium from bone, and an increased reabsorption of calcium in the kidney. The production of the final form of vitamin D, 1,25-dihydroxy vitamin D, is regulated by the need for calcium and phosphorus. Low serum calcium concentration stimulates the parathyroid gland to secrete parathyroid hormone, which in turn triggers the production of the "1,25--(OH).sub.2 D" in the kidney. 1,25--(OH).sub.2 D then directs the intestine to absorb calcium and phosphorus and the bone to mobilize calcium, and it stimulates renal reabsorption of calcium. These effects raise blood calcium to normal levels which in turn shut down parathyroid secretion, shutting down further production of 1,25--(OH).sub.2 D. Measurement of the level of 1,25-dihydroxy vitamin D in the blood is therefore an important diagnostic tool with respect to certain diseases (e.g. kidney failure, osteoporosis). It may also in the future provide useful research information.
Measurement of the levels in the blood of the precursor, 25-hydroxy vitamin D, has been carried out in the past by high performance liquid chromatography and by competitive protein binding assay. J. Eisman et al., 80 Anal Biochem. 298-305 (1977); J. Haddad et al., 33 J. Clin Endocr 992995 (1971). The disclosure of these articles and all other articles recited herein are incorporated by reference as if fully set forth herein. A protein used in the prior art competitive binding assay was the vitamin D transport protein, called "DBP" This protein has a strong preference for binding of 25--OH vitamin D as distinguished from vitamin D itself or 1,25--(OH).sub.2 D. R. Bouillion et al., 13 J. Steriod Biochem. 1029-1034 (1980).
There have also been prior art attempts to assay for 1,25--(OH).sub.2 vitamin D. D. Shigeharu et al., 116 Anal. Biochem. 211-222 (1981); J. Eisman et al., 176 Arch. Biochem Biophys. 235-243 (1976). These methods rely on competitive binding assay techniques or development of an antibody that binds to vitamin D metabolites (e.g. dihydroxycholecaliciferol). See generally H. Perry et al., 112 Biochem. Biophys. Res. Commun. 431-436 (1983); R. Bouillion et al., 41 Ann. Endocrin. 435-36 (1980); R. Bouillon, 26 Clin. Chem. 562-567 (1980); R. Bouillon, 66 Eur. J. Biochem., 285-291 (1976).
In such assays, mammalian blood serum or plasma is treated with an organic solvent that extracts vitamin D and its metabolites. The extract is then pre-purified on a column. The semi-purified 1,25--(OH).sub.2 D is then further purified by high performance liquid chromatography, yielding the purified 1,25--(OH).sub.2 D. During these steps there are usually losses of the 1,25--(OH).sub.2 D. To correct for these losses, the original plasma or serum extract has added to it a measured amount of radiolabeled 1,25--(OH).sub.2 D. After the final isolation and before actual measurement by binding assay, the radioactivity remaining in the isolated material is counted to allow computation of a recovery. This recovery is then used in the final calculation to correct for the losses of 1,25--(OH).sub.2 D during purification.
The isolated 1,25--(OH).sub.2 D from serum is then added to a mixture of radiolabeled 1,25--(OH).sub.2 D and either 1,25--(OH).sub.2 D receptor which is a protein that specifically binds 1,25--(OH).sub.2 D or an antibody raised to vitamin D metabolites. The unlabeled 1,25--(OH).sub.2 D in the serum will compete with the radiolabeled 1,25--(OH).sub.2 D. The degree to which the binding of labeled 1,25--(OH).sub.2 D is reduced by unlabeled 1,25--(OH).sub.2 D is used to construct a standard curve to determine the amount of 1,25--(OH).sub.2 D present in the sample.
The level of bound, labeled 1,25--(OH).sub.2 D is determined by absorbing the free or unbound labeled 1,25l--(OH).sub.2 D on dextran-coated charcoal. See e.g. J. Haddad et al., 33 J. Clin. Endocr. 992-995 (1971). As will be appreciated, these prior art assays require several days to complete and have many sources of possible error. They are also unduly costly. Therefore, there is need for a simple, rapid, relatively inexpensive, and accurate assay for 1,25--(OH).sub.2 D.